Solventless extraction process

ABSTRACT

The present invention provides a method for extracting lipids from microorganisms without using organic solvent as an extraction solvent. In particular, the present invention provides a method for extracting lipids from microorganisms by lysing cells and removing water soluble compound and/or materials by washing the lysed cell mixtures with aqueous washing solutions until a substantially non-emulsified lipid is obtained.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of priority under 35U.S.C. §119(e) from Provisional Patent Application Serial No.60/177,125,filed on Jan. 19, 2000. Provisional Patent Application Serial No.60/177,125 is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is directed to a process for extractinglipids from microorganisms without the use of any significant amount ofan organic solvent.

BACKGROUND OF THE INVENTION

[0003] A typical microorganism lipid manufacturing process, such asproduction of omega-3 highly unsaturated fatty acid, in particulardocosahexaenoic acid (DHA), involves growing microorganisms which arecapable of producing the desired lipid in a fermentor, pond orbioreactor, isolating the microbial biomass, drying it, and extractingintracellular lipids with an organic solvent, e.g., hexane. Generally,intracellular lipids of microorganisms are extracted after rupturing(i.e., lysing) the cells of the microorganisms. The extracted lipids arecan be further refined to produce a high purity and/or quality lipids.The microorganisms are generally isolated by first diluting thefermentation broth with water, and centrifuging the mixture to isolatemicroorganisms. When lipids are not extracted immediately or soon afterisolating the microorganisms, the isolated microorganisms are typicallydried, for example, on a drum dryer, and packaged, for example, invacuum-sealed bags, to prevent degradation of lipids.

[0004] Unfortunately, the drying process exposes the microorganisms toheat, which can damage, i.e., degrade the quality of, lipids if doneincorrectly. The vacuum-sealed bags may develop leaks, which can furtherdegrade the quality of the lipids due to exposure of the microorganismsto air. In addition, if the dried microorganisms are not treated with anantioxidant, lipids can be further degraded due to exposure to air, forexample, DHA may degrade due to oxidation by air. Furthermore, in somecases operators who are exposed to the dried microorganisms can developan allergic reaction creating a safety and/or health hazard tooperators.

[0005] Moreover, in an industrial scale production, the amount oforganic solvent used in lipid extraction typically requires a largeamount of volatile and flammable organic solvent, thereby creatinghazardous operating conditions. The use of organic solvent in theextraction process may necessitate using an explosion-proof oil recoverysystem, thereby adding to the cost of lipid recovery. Moreover, use ofan organic solvent in extracting lipids from microorganisms generate anorganic solvent waste stream requiring a proper method, which furtherincreases the overall production cost of lipid extraction.

[0006] Therefore, there is a need for a process for extracting lipidsfrom microorganisms which does not require the use of an organicsolvent. There is also a need for a lipid extraction process frommicroorganisms which does not require the expensive step of drying themicroorganisms.

SUMMARY OF THE INVENTION

[0007] The present invention provides a process for obtaining lipid frommicroorganisms comprising:

[0008] (a) lysing cells of the microorganisms to produce a lysed cellmixture;

[0009] (b) treating the lysed cell mixture to produce a phase separatedmixture comprising a heavy layer and a light layer;

[0010] (c) separating the heavy layer from the light layer; and

[0011] (d) obtaining the lipid from the light layer.

[0012] The lysed cell mixture may contain an emulsion, in which case theemulsion can be separated by centrifuging the lysed cell mixture. Theseparated lysed cell mixture comprises a heavy layer which containsaqueous solution and a light layer which contains lipids, which may beemulsified. The aqueous solution comprises solid cell materials whichresults from lysing cells. The light layer can be further washed with anaqueous washing solution until the lipid becomes substantiallynon-emulsified.

[0013] When the lipid extraction process of the present inventionincludes using microorganisms from a fermentation process, theextraction process can also include solubilizing at least part ofproteinaceous compounds in a fermentation broth, by adding a baseselected from the group consisting of hydroxides, carbonates,bicarbonates and mixtures thereof.

[0014] The process of the present invention can also include heating themicroorganisms to temperature of at least about 50° C.

[0015] Preferably, the microorganisms are capable of growth at salinitylevel of less than about 12 g/L of sodium chloride, more preferably lessthan about 5 g/L of sodium chloride and most preferably less than about3 g/L of sodium chloride.

[0016] Preferably, the microorganisms comprise at least about 30% byweight of lipid, more preferably at least about 35% by weight, and mostpreferably at least about 40%. Alternatively at least about 30% of thelipid is docosahexaenoic acid, preferably at least about 35%, and morepreferably at least about 40%.

[0017] In one particular aspect of the present invention themicroorganisms are capable of producing at least about 0.1 grams perliter per hour of docosahexaenoic acid, more preferably at least about0.2 g/L/h, still more preferably at least about 0.3 g/L/h, and mostpreferably at least about 0.4 g/L/h.

[0018] In another aspect of the present invention, the microorganism isselected from the group consisting of algae, fungi, bacteria andprotist. Preferably, the microorganisms are of the orderThraustochytriales. More preferably the microorganisms are selected fromthe genus Thraustochytrium, Schizochytrium and mixtures thereof And mostpreferably, the microorganisms are selected from the group consisting ofmicroorganisms having the identifying characteristics of ATCC number20888, ATCC number 20889, ATCC number 20890, ATCC number 20891 and ATCCnumber 20892, mutant strains derived from any of the foregoing, andmixtures thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is a flow diagram of one embodiment of a solventlessextraction process of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention is directed to a process for extracting,recovering, isolating or obtaining lipids from microorganisms. Theprocess of the present invention is applicable to extracting a varietyof lipids from a variety of microorganisms, for example, extractinglipids containing omega-3 highly unsaturated fatty acids, such asdocosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and/ordocosapentaenoic acid (DPA), in particular lipids containing arelatively large amount of DHA, from microorganisms producing the sameand extracting lipids containing arachidonic acid from microorganismsproducing the same. Exemplary microorganisms which produce a relativelylarge amount of omega-3 highly unsaturated fatty acids are disclosed incommonly assigned U.S. Pat. Nos. 5,340,594 and 5,340,742, both issued toBarclay, and exemplary microorganisms which produce a relatively largeamount of arachidonic acid are disclosed in commonly assigned U.S. Pat.No. 5,583,019, issued to Barclay. All of the above disclosed patents areincorporated herein by reference in their entirety.

[0021] For the sake of brevity, however, this detailed description ofthe invention is presented for purposes of convenience and illustrationfor the case of extracting lipids comprising omega-3 highly unsaturatedfatty acid from microorganisms producing the same, in particularextracting lipids from microorganisms which produce a relatively highamount of DHA. It is to be understood, however, that the invention as awhole is not intended to be so limited, and that one skilled in the artwill recognize that the concept of the present invention will beapplicable to other microorganisms producing a variety of lipidcompositions in accordance with the techniques discussed herein. Thesemicroorganisms include microorganisms, such as fungi, protist, algae andbacteria, which produce a variety of lipids, such as phospholipids; freefatty acids; esters of fatty acids, including triglycerides of fattyacids; sterols; pigments (e.g., carotenoids and oxycarotenoids) andother lipids, and lipid associated compounds such as phytosterols,ergothionine, lipoic acid and antioxidants including beta-carotene andtocopherol. Exemplary lipids include, but are not limited to,arachidonic acid, stearidonic acid, cholesterol, desmesterol,astaxanthin, canthaxanthin, and n-6 and n-3 highly unsaturated fattyacids such as eicosapentaenoic acid, docosapentaenoic acid anddocosahexaenoic acid. For the sake of brevity, unless otherwise stated,the term “lipid” refers to lipid and/or lipid associated compounds.Other lipids and microorganisms which may be suitable for use in theinstant invention will be readily apparent to those skilled in the art.

[0022] Typical microbial lipid (in particular an oil containing anomega-3 highly unsaturated fatty acid such as DHA) manufacturingprocesses involve growing microorganisms which produce DHA in afermentor, isolating the microorganisms, and extracting theintracellular lipids with organic solvent, e.g., hexane. The extractedlipid is generally further refined to produce a high purity and/orquality lipid. The isolation of microorganisms involves diluting thefermentation broth with water and centrifuging the mixture to isolatemicroorganisms. When lipids are not extracted immediately or soon afterisolating the microorganisms, the isolated microorganisms are typicallydried, for example, on a drum dryer, and sealed in a package, e.g., invacuum-sealed bags, to prevent degradation of lipids. Unfortunately, thedrying process exposes the microorganisms to heat, which can damage,i.e., degrade the quality of, the lipid if done incorrectly. The packagemay develop leaks, which can further degrade the quality of the lipids.Furthermore, if the dried microorganisms are not treated with anantioxidant, the exposure of microorganisms to air can further degradelipids.

[0023] Recovering the crude oil directly from the fermentation brothavoids these problems. Avoiding the organic solvent extraction stepreduces manufacturing costs and also eliminates operator exposure to thedried microorganisms, which can cause an allergic response in someindividuals.

[0024] The present invention provides a method for obtaining lipids frommicroorganisms using a substantially organic solvent free extractionprocess, i.e., a “solventless” extraction process. The term “solventlessextraction process” refers to an extraction process which when anaqueous solvent is used, the aqueous solvent comprises less than about5% of an organic solvent, preferably less than about 4%, more preferablyless than about 2%, and most preferably less than 1%. The process of thepresent invention can include obtaining or isolating microorganisms,preferably from a fermentation process. In contrast to the currentmethods, the process of the present invention does not require a dryingstep prior to the extraction process. Thus, processes of the presentinvention are applicable to extracting lipids from a microbial biomasscontaining at least about 10% by weight entrained water, preferably atleast about 20%, more preferably at least about 30%, and most preferablyat least about 50%. When the microorganisms are obtained from afermentation process, the process of the present invention can includeadding a base to the fermentation broth to dissolve any proteinaceouscompound that may be present in the broth. A “base” refers to anycompound whose pKa is greater than that of water. The base should bestrong enough to hydrolyze at least a portion of proteinaceous compoundsthat may be present in the broth. Bases which are useful forsolubilizing proteins are well known to one of ordinary skill in the artof chemistry. Exemplary bases which are useful in the processes of thepresent invention include, but are not limited to, hydroxides,carbonates and bicarbonates of lithium, sodium, potassium, calcium, andmagnesium carbonate.

[0025] The process of the present invention can also include rupturingor lysing the cells of microorganisms to release the lipids which arepresent within the cells. Cells can be lysed using any of the knownmethods including chemical; thermal; mechanical, including, but notlimited to, french press, mills, ultrasonication, and homogenization;and combinations thereof. In a thermal lysing of cells, the fermentationbroth containing microorganisms are heated until cells, i.e., cellwalls, of microorganisms degrade or breakdown. Typically, thefermentation broth is heated to a temperature of at least about 50° C.,preferably at least about 75° C., more preferably to at least about 100°C., and most preferably to at least about 130° C. Thermally lysing thecell walls of microorganisms is particularly useful for microorganismswhose cell walls are composed of proteins.

[0026] Heating the broth also denatures proteins and helps solubilizeorganic materials, including proteins. Heating of the fermentation brothstep can be achieved by any of the known methods, including the use ofan in-line heat exchanger, and preferably by sparging steam into thefermentor and maintaining the broth at a desired temperature for lessthan about 90 minutes, preferably less than about 60 minutes, and morepreferably less than about 30 minutes.

[0027] The solventless extraction process of the present invention canalso include at least partially separating the broth from lipids.Typically, this is achieved by centrifuging, e.g., by passing the broththrough a stacked-disc centrifuge, and collecting lipids as an emulsionphase. Centrifuging the mixture results in a two phase mixturecomprising a heavy layer and a light layer. Typically, the heavy layeris an aqueous phase, which contains the majority of cellular debris. Thelight layer which contains emulsified lipids is then diluted with water,again separated into two phase mixture and the light layer is againisolated. This dilution with water, separation and isolation processes(i.e., washing process) can be achieved continuously by feeding waterand removing the heavy layer throughout the process or it can beconducted in discreet steps. The washing process is generally repeateduntil a non-emulsified lipid layer is obtained. It is believed that theoil-water interface of the emulsion is stabilized by residual cellulardebris which is removed by the washing process. During the washingprocess, the successive amount of water added is reduced to increase thelipid content. While reducing the amount of feed water too quickly canresult in loss of lipids to the aqueous phase, reducing the amount offeed water too slowly results in an inefficient washing process. One canreadily determine an appropriate rate of feed water reduction byobserving or analyzing the separated aqueous layer. Generally, the lipidlayer, i.e., the light layer, is colored; therefore, in many cases onecan determine an appropriate rate of feed water reduction by simplyanalyzing or observing the color of the aqueous layer which is separatedfrom the lipid layer.

[0028] The isolated lipid can be further refined using a process similarto that used to refine standard vegetable oils. Briefly, the lipidrefining process generally involves hydrating phospholipids by addingphosphoric acid to the lipid followed by adding sodium hydroxide toneutralize free fatty acids. These compounds are removed viacentrifugation. This is then followed by a water wash step to furtherremove any remaining amounts of hydrated phospholipids (“gums”) andneutralized fatty acids (“soapstock”) in the lipid. The resulting lipidis bleached using Trysil™ and a standard bleaching clay. Citric acid isalso added to remove divalent metal ions by chelation. The Trysil™ andbleaching clay are then removed via filtration to produce refined lipid.The bleached lipid can be cold filtered to remove high melting pointcompounds that may be present in the lipid; however, this step isgenerally seldom required.

[0029] The resulting lipid can be further refined by removing any lowmolecular weight components that may be present. Typically, thesecomponents are removed by sparging with steam at high temperatures,under high vacuum. This process also destroys any peroxide bonds whichmay be present and reduces or removes off odors and helps improve thestability of the oil. An antioxidant may then be added to the resultingdeodorized lipid to improve product stability.

[0030] Prior to the refining process, the isolated lipid can bewinterized to remove high melting compounds, such as saturated fattyacids. The winterization process generally involves dissolving theisolated lipid in an organic solvent, e.g., hexane, cooling theresulting organic solution, and filtering the solution to remove thehigh melting point components of the lipid or stearine phase. Thewinterization process generally produces a clear lipid, especially whenthe isolated lipid is cloudy or opaque.

[0031] While, the process of the present invention can include isolatingmicroorganisms from a fermentation process, one of the advantages of thepresent invention is that it allows fermentation of microorganisms andisolation of lipids to be carried out in a single vessel.

[0032] For example, after the fermentation, one can add base to thefermentation vessel and heat the mixture to lyse cells. After separatingthe phase into a heavy layer and a light layer, the light layer can betransferred to another vessel for further processing or the heavy layercan be removed from the fermentation vessel, for example, by drainingthrough the bottom of the fermentation vessel, and the remaining lightlayer can be further processed within the same fermentation vessel.

[0033] Additional objects, advantages, and novel features of thisinvention will become apparent to those skilled in the art uponexamination of the following examples thereof, which are not intended tobe limiting.

EXAMPLES

[0034] Process reproducibility was characterized by producing threesamples of fully refined oil using crude oil from the new solventlessextraction process. A hexane-extracted sample was also fully refined toserve as a control. The fermentation, extraction and oil isolation stepswere performed at a large scale, while the oil refining studies wereperformed at a small scale.

[0035] The fully refined oil samples were analyzed to demonstrateprocess reproducibility.

[0036] Fermentation:

[0037] A single F-Tank batch (˜1,200 gallons) was used to generate thestarting broth for the three solventless extraction processes. The batch(#F99202) was allowed to run for 94 hours, while controlling the glucoselevels at 13 g/L, after which time the corn syrup feed was terminated.Residual glucose levels dropped to <5 g/L four hours later. Thisresulted in a final age of 98 hours. The final broth volume was 958gallons. The final yield was 146 g/L. Both in-process contaminationchecks and a thorough analysis of a final broth sample failed to showany signs of contamination.

[0038] Hexane-Extracted Control Sample:

[0039] A small aliquot of broth from batch #F99202 was drum-dried andextracted with hexane to serve as a control sample. The biomassintermediate (DHAINT Lot #9F0067A) was recovered using a 66 ft²double-drum dryer. Analysis of this lipid is shown in Table 1. TABLE 1Analysis of DHAINT Lot #9F0067A. Parameter Value DHA Content (FAMEbasis) 35.7% Oil Content 62.7% Peroxide Value (meq/kg) 2.6 Total PlateCount (cfu/g) <50 DHA Content* 20.3% FAME Content 56.9%

[0040] Solventless Extraction Process:

[0041] Crude oil was obtained by treating three 400-gallon aliquots(approx.) of broth in batch #F99202. Each 400-gallon aliquot from theF-Tank batch was processed separately, starting with the caustic/heattreatment steps. Each aliquot was treated with 20 grams of 45% KOH perliter and heated to 130° C. for about 30 minutes by passing streamthrough the fermentation broth. The crude oil was recovered from thetreated broth using a commercial-scale Westfalia HFA-100 stacked-disccentrifuge. Summary results for various process parameters are reportedin Table 2, and the final crude oil analysis results are shown in Table3. TABLE 2 Process Data from the Solventless Extraction Process. SFE-1SFE-2 SFE-3 Broth Treatment Volume of Broth Processed 288 gal 288 gal258 gal Final Treated pH  7.5  8.0  8.7 Final Volume After HeatTreatment 388 gal 398 gal 308 gal Volume Increase From Condensate  34.7% 38.2%  19.4% 1^(st) Pass Emulsion Total Volume (gal) 180 133 149 Est.Oil Concentration (w/w)  12.0%  24.5%  16.1% Apparent Density (g/mL) 0.986  0.991  0.999 Oil Isolation Total Crude Oil Recovered (lb) 182165 174 DHAOIL Lot Number Assigned 9F0001A 9F0002A 9F0003A

[0042] TABLE 3 Analysis of Lots of DHA from the Solventless ExtractionProcess. Parameter 9F0001A 9F0002A 9F0003A DHA Content (% FAME) 39.0%38.6% 39.2% Peroxide Value (meq/kg) 4.6 1.8 2.0 Acid Value (mg KOH/g)N/D N/D N/D Moisture Content N/D N/D N/D

[0043] Refining:

[0044] A sample from each aliquot of crude oil was winterized, refined,bleached and deodorized at a small scale, as was a sample of the crudeoil from the hexane-extracted control. Miscellaneous process data fromthese small scale experiments is shown in Table 4, including recoveryefficiencies for the various processing steps. While it is difficult toread too much into recovery efficiencies for bench-scale processes, aslosses tend to be disproportionately large, the values listed in Table 4show that values for the solventless-extracted samples tend to bracketthe values measured for the hexane-extracted control, with the oneexception being the winterization step. While the recovery efficiencyduring the winterization step for the hexane control was lower thanthose observed for the other three samples, this difference isinsignificant from a statistical perspective. The high losses during thewinterization step caused the overall recovery efficiency for thehexane-control sample to be lower as well. The lower yield would not beexpected to have a significant impact on the overall quality of the oil.All in all, differences in the processing of the various oil sampleswere minimal. TABLE 4 Miscellaneous Process Data from the Oil RefiningSteps. HEX-1 SFE-1 SFE-2 SFE-3 Processing Conditions MiscellaConcentration 45.0% 52.9% 52.8% 45.0% Steam Sparge Rate  3.4%  3.4% 2.5%  2.2% Recovery Efficiencies Winterization 80.6% 92.3% 87.7% 85.5%Refining 89.4% 84.8% 91.8% 95.0% Water Wash 90.6% 94.5% 95.8% 81.2%Bleaching 86.1% 89.2% 87.3% 84.1% Deodorization 97.4% 96.1% 97.2% 97.5%Packaging 88.2% 89.7% 89.3% 95.8% Overall 48.2% 56.9% 58.5% 51.8% FinalProduct Lot Number 9F0009A 9F0010A 9F0011A 9F0012A

[0045] Fully refined oil samples from the three solventless extractionruns, and the hexane-extracted control, were analyzed and the resultsare shown in Table 5. Also shown are the corresponding releasespecifications for each parameter.

[0046] A sample of the starting crude oil from the solventlessextraction run was also analyzed for iron content. The iron content ofthis sample (DHAOIL Lot #9F0002P) was 0.08 ppm. The concentration of theother trace metals was all below their respective detection limits.TABLE 5 QC Results for RBD Oil from the Solventless Extraction Process.Hexane Solventless Extraction Run ID # HEX-1 SFE-1 SFE-2 SFE-3 DHALIP-NSLot # 9F0009A 9F0010A 9F0011A 9F0012A Peroxide Value 0.28 0.69 0.35 0.34(meq/kg) Acid Value 0.17 0.11 0.57 0.24 (mg KOH/g) Moisture & Volatiles0.00% 0.06%** 0.00% 0.00% Trace Metals (ppm) Lead <0.20 <0.20 <0.20<0.20 Arsenic <0.20 <0.20 <0.20 <0.20 Iron 0.22 0.21 0.56*** 0.02 Copper<0.05 <0.05 <0.05 <0.05 Mercury <0.20 <0.20 <0.20 <0.20 DHA (% FAME)36.9 37.3 37.0 37.7 DHA (mg/g oil) 342 345 343 351 Hexane (ppm) <3 <3 <3<3

[0047] Shown in Table 6 is a more direct comparison of the averageanalysis results for the three samples from the solventless extractionprocess versus those for the hexane control. TABLE 6 Comparison ofAverage Values. Hexane Solventless Extraction Parameter Control Mean StdDev CV % Diff Peroxide Value 0.28 0.46 0.20 43.3% 64.3% (meq/kg) AcidValue 0.17 0.19* 0.06 33.3% 11.2% (mg KOH/g) Moisture & 0.00% 0.02%0.03%  173% ND Volatiles Trace Metals (ppm) Lead <0.20 <0.20 N/A N/A0.0% Arsenic <0.20 <0.20 N/A N/A 0.0% Iron 0.22 0.26 0.27  104% 18.2%Copper <0.05 <0.05 N/A N/A 0.0% Mercury <0.20 <0.20 N/A N/A 0.0% DHAContent 36.9% 37.3% 0.4%  0.9% 1.1% (% FAME) DHA Content 342 346 4  1.2%1.2% (mg/g) Hexane (ppm) <3 <3 N/A N/A 0.0%

[0048] The results from this experiment clearly demonstrate that thesolventless extraction process is both reproducible and lipids fromsolventless extraction are relatively indistinguishable from the lipidsobtained from hexane extraction process in terms of process performanceand product quality. The final product from the solventless extractionprocess is substantially equivalent to lipids from a currenthexane-based extraction process, as determined by similarities betweenthe fatty acid and sterol profiles of the product from these twoprocesses.

[0049] The present invention, in various embodiments, includescomponents, methods, processes, systems and/or apparatus substantiallyas depicted and described herein, including various embodiments,subcombinations, and subsets thereof. Those of skill in the art willunderstand how to make and use the present invention after understandingthe present disclosure. The present invention, in various embodiments,includes providing devices and processes in the absence of items notdepicted and/or described herein or in various embodiments hereof,including in the absence of such items as may have been used in previousdevices or processes, e.g., for improving performance, achieving easeand/or reducing cost of implementation.

[0050] The foregoing discussion of the invention has been presented forpurposes of illustration and description. The foregoing is not intendedto limit the invention to the form or forms disclosed herein. Althoughthe description of the invention has included description of one or moreembodiments and certain variations and modifications, other variationsand modifications are within the scope of the invention, e.g., as may bewithin the skill and knowledge of those in the art, after understandingthe present disclosure. It is intended to obtain rights which includealternative embodiments to the extent permitted, including alternate,interchangeable and/or equivalent structures, functions, ranges or stepsto those claimed, whether or not such alternate, interchangeable and/orequivalent structures, functions, ranges or steps are disclosed herein,and without intending to publicly dedicate any patentable subjectmatter.

1. A process for obtaining lipid from microorganisms comprising: a)lysing cells of the microorganisms to produce a lysed cell mixture; b)treating said lysed cell mixture to produce a phase separated mixturecomprising a heavy layer and a light layer, wherein said heavy layercomprises an aqueous solution and said light layer comprises said lipid;c) separating said heavy layer from said light layer; and d) obtainingsaid lipid from said light layer.
 2. The process of claim 1, whereinsaid step (b) comprises centrifuging said lysed cell mixture.
 3. Theprocess of claim 2, wherein said light layer comprises an emulsifiedlipid.
 4. The process of claim 3 further comprising: e) adding anaqueous extraction solution to said light layer of step (c); and f)repeating said steps (b), (c) and (e) until said lipid becomessubstantially non-emulsified prior to said step (d).
 5. (Cancelled) 6.(Cancelled)
 7. (Cancelled)
 8. The process of claim 7 further comprisingadding a base to a fermentation broth.
 9. (Cancelled)
 10. The process ofclaim 7 further comprising solubilizing at least part of proteinaceouscompounds in a fermentation broth.
 11. The process of claim 1, whereinsaid step (a) comprises heating said microorganisms to temperature of atleast about 50° C.
 12. The process of claim 1, wherein saidmicroorganism is capable of growth at salinity level of less than about12 g/L of sodium chloride.
 13. (Cancelled)
 14. The process of claim 1,wherein said microorganism is selected from the group consisting ofalgae, fungi, bacteria and protist.
 15. The process of claim 14, whereinsaid microorganisms comprise microorganisms of the orderThraustochytriales.
 16. (Cancelled)
 17. (Cancelled)
 18. The process ofclaim 1, wherein said microorganisms are capable of producing at leastabout 0.1 grams per liter per hour of docosahexaenoic acid.
 19. Theprocess of claim 1, wherein at least about 30% of said lipid isdocosahexaenoic acid.
 20. A process for obtaining lipids frommicroorganisms comprising: a) growing said microorganisms in afermentation medium to produce a fermentation broth; b) solubilizing atleast a part of any proteins present in said fermentation broth; c)lysing cells of said microorganisms to produce a lysed cell mixture; d)treating said lysed cell mixture to produce a phase separated mixturecomprising a heavy layer and a light layer, wherein said heavy layercomprises an aqueous solution and said light layer comprises emulsifiedlipids; e) separating said heavy layer from said light layer; and f)obtaining said lipids from said light layer.
 21. The process of claim20, wherein said step of dissolving proteins comprises contacting saidfermentation broth with a base.
 22. (Cancelled)
 23. The process of claim20, wherein said step of lysing cells comprises heating saidmicroorganisms to a temperature of at least about 50° C.
 24. The processof claim 20, wherein said step of producing the phase separated mixturecomprises centrifuging said lysed cell mixture.
 25. The process of claim20, wherein said step of obtaining said lipids from said light layercomprises: (A) adding an aqueous washing solution to said light layer;(B) separating said aqueous washing solution from said light layer; and(C) repeating said steps (A) and (B) until said lipid becomessubstantially non-emulsified. 26-34. (Cancelled)
 35. A process forobtaining lipids from microorganisms comprising: a) growing saidmicroorganisms in a fermentation medium to produce a fermentation broth;b) contacting said fermentation broth with a base to dissolve at least apart of any proteins present in said fermentation broth; c) increasingthe temperature of said fermentation broth to at least about 50° C. tolyse cells of said microorganisms to produce a lysed cell mixture; d)separating substances of different densities from said lysed cellmixture to produce a phase separated mixture comprising a heavy layerand a light layer, wherein said heavy layer comprises an aqueoussolution and said light layer comprises emulsified lipids; e) removingsaid heavy layer from said phase separated mixture; f) adding an aqueouswashing solution to said light layer; g) separating substances ofdifferent densities from said mixture of step (f); h) removing saidheavy layer from said phase separated mixture; and i) repeating saidsteps (f)-(h) until said lipid becomes substantially non-emulsified. 36.The process of claim 35, wherein said base is selected from the groupconsisting of hydroxides, carbonates, bicarbonates, and mixturesthereof.
 37. The process of claim 35, wherein said step of producing thephase separated mixture comprises centrifuging said lysed cell mixture.38-46. (Cancelled)